The present invention relates to compounds which have metallo-xcex2-lactamase inhibitory characteristics. The invention also relates to methods of preparing, pharmaceutical compositions and uses of the compounds.
Metallo-xcex2-lactamases are bacterial enzymes which confer resistance to virtually all clinically relevant xcex2-lactam antibiotics, including carbapenems and jeopardize the future use of all such agents. The increased treatment of infections with carbapenems and other xcex2-lactam antibiotics may lead to the proliferation of clinical bacterial strains which are able to produce metallo-xcex2-lactamases and thus resist the effects of xcex2-lactam antibiotics. In fact, metallo-xcex2-lactamases have now been identified in a number of pathogenic bacterial species including Bacillus cereus, Bacteroides fragilis, Aeromonas hydrophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Stenotrophomonas maltophilia, Shigella flexneri, Legionella gormanii, Chryseobacterium meningosepticum, Chryseobacterium indologenes, Acinetobacter baumannii, Citrobacter freundii, and Aeromonas veronii. 
Accordingly, there is an increasing need for agents which when combined with a xcex2-lactam antibiotic, e.g. imipenem, will restore the effectiveness of the xcex2-lactam antibiotics and which are at the same time relatively free from undesirable side effects.
WO 98/17639, 97/30027, 98/40056, 98/39311 and 97/10225 teach certain beta-thiopropionyl-amino acid derivatives and their use as inhibitory agents against metallo-xcex2-lactamases. Goto et. al., Biol. Pharm. Bull. 20, 1136 (1997), Payne et. al., FEMS Microbiology Letters 157, 171 (1997), Payne et al., Antimicrob. Agents Chemother. 41, 135 (1997), Page et. al., Chem. Commun. 1609 (1998) and Page et al., Biochem. J. 331, 703 (1998) also disclose certain thiols and thioesters as metallo-xcex2-lactamase inhibitors. Additionally, Toney et al., Chemistry and Biology 5, 185 (1998), Fastrez et al., Tetrahedron Lett. 36, 9313 (1995), Schofield et al., Tetrahedron 53, 7275 (1997), Schofield et. al., Bioorg. and Med. Chem. Lett. 6, 2455 (1996) and WO 97/19681 disclose other metallo-xcex2-lactamase inhibitors. However, the above noted references do not teach the compounds of the instant invention.
Other references which disclosed the general state of the art are Bush et al., Antimicrob. Agents Chemother. 41, 223 (1997); Livermore, D. M. J. Antimicrob. Chemother. 1998, 41 (Suppl. D), 25; Bush, K. Clin. Infect. Dis. 1998, 27 (Suppl 1), S48; Livermore, D. M. J. Antimicrob. Chemother. 1997, 39, 673 and Payne, D. J. J. Med. Microbiol. 1993, 39, 93.
This invention relates to novel substituted succinic acid metallo-xcex2-lactamase inhibitors, which are useful potentiators of xcex2-lactam antibiotics. Accordingly, the present invention provides a method of treating bacterial infections in animals or humans which comprises administering, together with a xcex2-lactam antibiotic, a therapeutically effective amount of a compound of formula I: 
including pharmaceutically acceptable salts, prodrugs, anhydrides, and solvates thereof, wherein:
M1 and M2 are independently selected from:
(a) Hydrogen,
(b) Pharmaceutically acceptable cation,
(c) Pharmaceutically acceptable esterifying group; and
(d) A negative charge;
R1 and R2 are independently selected from the following:
(a) Hydrogen, provided that R1 and R2 are not hydrogen at the same time;
(b) a C1 to C16 straight, branched or unsaturated alkyl group substituted with 0 to 2 Rq groups and substituted with 0 to 3 Rx groups and optionally interrupted by one of the following O, S, SO2, xe2x80x94C(O)xe2x80x94,
(c) xe2x80x94C(O)xe2x80x94NRaxe2x80x94, xe2x80x94CO2xe2x80x94;
(c) a group of the formula: 
xe2x80x83wherein
xe2x80x94Axe2x80x94 represents a single bond, C1 to C8 straight, branched or unsaturated alkyl group optionally substituted with 1 to 2 Rx groups and optionally interrupted by one of the following O, S, SO2, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94NRaxe2x80x94, xe2x80x94CO2xe2x80x94; 
represents:
(1) a C6 to C14 aryl group;
(2) a C3 to C10 alicyclic group;
(3) a C3 to C14 heteroaryl group, which contains 1 to 3 heteroatoms, 0 to 3 of which heteroatoms are nitrogen and 0 to 1 of which are oxygen or sulfur;
(4) a C3 to C10 heterocyclic group, which contains 1 to 2 heteroatoms, 0 to 1 of which heteroatoms are nitrogen, and 0 to 2 of which are oxygen or sulfur; or
(d) a group of the formula: 
xe2x80x83wherein:
xe2x80x94Axe2x80x94 is as defined above;
Axe2x80x2 is a single bond, O, S, or a C1 to C6 straight, branched or unsaturated alkyl group optionally substituted with 1-2 Rx groups and optionally interrupted by one of the following groups O, S, SO2, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94NRaxe2x80x94, xe2x80x94CO2xe2x80x94; 
are independently selected from:
(1) a C6 to C10 aryl group;
(2) a C3 to C8 alicyclic group;
(3) a C2 to C9 heteroaryl group, which contains 1 to 3 heteroatoms, 0 to 3 of which heteroatoms are nitrogen and 0 to 1 of which are oxygen or sulfur;
(4) a C3 to C8 heterocyclic group, which contains 1 to 2 heteroatoms, 0 to 1 of which heteroatoms are nitrogen, and 0 to 2 of which are oxygen or sulfur;
provided that at least one Rq group is present in R1 or R2 and that when more than one Rq is present the total number of cationic nitrogen atoms does not exceed 8; the total number of cationic nitrogen atoms can be charged balanced by M1 and/or M2 or by M1 and/or M2 in combination with an appropriate number of Yxe2x88x92; wherein:
Rq is xe2x80x94Exe2x80x94Q+Yxe2x88x92;
Yxe2x88x92 is a pharmaceutically acceptable anionic group;
E is xe2x80x94(CH2)mxe2x80x94Xxe2x80x94(CH2)nxe2x80x94;
m is 0 to 6;
n is 0 to 6 (but when E is attached to an aromatic ring n is 1-6);
X is a bond, O, S, SO2, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94N(Ra)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94 or xe2x80x94Cxe2x89xa1Cxe2x80x94, provided that when X is O, S, xe2x80x94C(O)xe2x80x94N(Ra)xe2x80x94 or xe2x80x94C(O)Oxe2x80x94, then n is 2 to 6
and Q+, attached to the (CH2)n terminus of E is:
(1) a cationic group selected from the following: 
xe2x80x83wherein:
Ru and Rv are independently hydrogen or C1-6 alkyl optionally substituted with 1 to 2 Ry;
Rw is hydrogen or C1-6 alkyl optionally substituted with 1 to 2 Rx;
Ru and Rv when bonded to the same nitrogen atom may together be a C3-6 alkyl radical, which when taken together with the intervening atoms form a ring;
Two Ru groups on separate nitrogen atoms may together comprise a C2-5 alkyl radical, which when taken together with the intervening atoms form a ring;
Ru, Rv and Rw when bonded to the same nitrogen atom may together form a C6-10 tertiary alkyl radical, which with N+ forms a bicyclic ring;
(2) A dicationic group: 
xe2x80x83wherein:
E1 is xe2x80x94(CH2)pxe2x80x94Zxe2x80x94(CH2)rxe2x80x94;
p and r are independently 1 to 4;
Z is a bond, O, S, SO2, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)Oxe2x80x94**, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94Cxe2x89xa1Cxe2x80x94, or 
Provided that when Z is O or S, p is 2 to 4 and r is 2 to 4 and when Z is 
xe2x80x83or xe2x80x94C(O)Oxe2x80x94**, r is 2 to 4;
wherein ** denotes the atom which is bonded to the xe2x80x94(CH2)rxe2x80x94 moiety of E1 above;
Q1 is selected from the following: 
Q2 is selected from the following: 
Ru, Rv and Rw are independently selected and defined as above,
And in addition, in the case where two Ru groups on separate nitrogen atoms are joined to form a ring as defined above, two Rv groups on the same two separate nitrogen atoms may also comprise a C1-5 alkyl radical to form together with the intervening atoms a bicyclic ring; an example of such is: 
(3) A tricationic group selected from the following: 
xe2x80x83wherein:
Each E1 is as defined above, but selected independently;
Each Q1 is as defined above, but selected independently;
Each Q2 is as defined above, but selected independently;
Ru, Rv and Rw are defined as in the definition of Q+ item (2) above and selected independently; or
(4) A tetracationic group selected from the following: 
xe2x80x83wherein:
Each E1 is as defined above, but selected independently;
Each Q1 is as defined above, but selected independently;
Each Q2 is as defined above, but selected independently;
Ru, Rv and Rw are defined as in the definition of Q+ item (2) above and selected independently;
where each Rx is independently selected from the group consisting of:
(a) F, Cl, Br, I,
(b) CF3,
(c) ORb,
(d) CN,
(e) xe2x80x94C(O)xe2x80x94Rc,
(f) xe2x80x94S(O2)xe2x80x94Rf,
(g) xe2x80x94C(O)xe2x80x94ORa 
(h) xe2x80x94Oxe2x80x94C(O)xe2x80x94Rc,
(i) xe2x80x94Sxe2x80x94Rb,
(j) xe2x80x94N(Ra)xe2x80x94C(O)xe2x80x94Rc, 
(q) xe2x80x94N(Ra)xe2x80x94C(O)xe2x80x94ORf,
(r) xe2x80x94S(O)xe2x80x94Rf,
(s) xe2x80x94N(Ra)xe2x80x94S(O2)xe2x80x94Rf,
(t) NO2, and
(u) C1 to C8 straight, branched or unsaturated alkyl optionally substituted with one of the substituents (a) through (t) above;
(v) xe2x80x94CH2-aryl wherein the aryl is optionally substituted with one of the substituents (a) through (t) above;
or two adjacent Rx groups on an aromatic ring may consist of the following divalent moiety, xe2x80x94Oxe2x80x94CH2xe2x80x94Oxe2x80x94; wherein:
Ra is H, C1 to C6 alkyl optionally substituted with Ry;
Rb is H, C1 to C6 alkyl optionally substituted with Ry, CH2-aryl, or aryl, said aryls optionally substituted with 1-2 Ry groups;
Rc is H, C1 to C6 alkyl optionally substituted with Ry, CF3, or aryl, said aryl optionally substituted with 1-2 Ry groups;
Rd and Re are independently hydrogen, C1 to C4 alkyl optionally substituted with Ry, or Rd and Re taken together may represent a 3 to 5-membered alkyl radical to form a ring, or Rd and Re taken together may represent a 2 to 4-membered alkyl radical interrupted by O, S, SO or SO2 to form a ring;
Rf is C1 to C6 alkyl optionally substituted with Ry, or aryl, said aryl optionally substituted with 1-2 Ry groups; and
Ry is xe2x80x94OH, xe2x80x94OCH3, OCONH2, OCOCH3, CHO, COCH3, CO2CH3, CONH2, CN, SOCH3, SO2CH3, SO2NH2, F, Cl, Br, I or CF3.
The invention is intended to include all of the isomeric forms of the compounds of formula I, including racemic, enantiomeric and diastereomeric forms.
The invention is described herein in detail using the terms defined below unless otherwise specified.
The term xe2x80x9calkylxe2x80x9d refers to a monovalent alkane (hydrocarbon) derived radical containing from 1 to 16 carbon atoms unless otherwise defined. It may be straight or branched. Preferred alkyl groups include methyl, ethyl, propyl, isopropyl, butyl and hexyl. When substituted, alkyl groups may be substituted with up to 3 substituent groups selected from Rx, as defined, and up to 2 substituent groups selected from Rq, as defined, at any available point of attachment. When the alkyl group is said to be substituted with an alkyl group, this is used interchangeably with xe2x80x9cbranched alkyl groupxe2x80x9d. When the alkyl chain is interrupted by a group, e.g. O, this may occur between any two saturated carbons of the alkyl chain.
The term unsaturated alkyl refers to xe2x80x9calkenylxe2x80x9d or xe2x80x9calkynylxe2x80x9d. The term xe2x80x9calkenylxe2x80x9d refers to an unsaturated alkyl such as a hydrocarbon radical, straight or branched containing from 2 to 16 carbon atoms and at least one carbon to carbon double bond. Preferred alkenyl groups include propenyl, hexenyl and butenyl. The term xe2x80x9calkynylxe2x80x9d refers to an unsaturated alkyl such as a hydrocarbon radical straight or branched, containing from 2 to 16 carbon atoms and at least one carbon to carbon triple bond. Preferred alkynyl groups include propynyl, hexynyl and butynyl.
The term xe2x80x9calicyclicxe2x80x9d refers to non-aromatic monocyclic or bicyclic C3-C10 hydrocarbons, including unsaturated, which can be substituted with 0-3 groups of Rx. Examples of said groups include cycloalkyls such as cyclohexyl, cyclopentyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1]hepta-2,5-dienyl, bicyclo[2.2.2]octyl, bicyclo[2.2.2]octa-2,5-dienyl.
The term xe2x80x9calkylidenexe2x80x9d refers to an alkyl group which is attached through two bonds on the same carbon atom of the alkyl group to a single attachment atom Examples of said groups include methylene, ethylidene, isopropylidene and the like.
Examples of when Rd and Re are taken together along with the adjacent nitrogen atom to represent a 3 to 5 membered alkyl radical forming a ring or a 2 to 4 membered alkyl radical interrupted by O, S, SO, SO2, to form a ring are pyrrolidinyl, piperidinyl, morpholinyl and the like.
The term xe2x80x9cheterocyclicxe2x80x9d refers to a monocyclic non-aromatic moiety containing 3-8 ring atoms or a bicyclic non-aromatic moiety containing 6-10 ring atoms, at least one of which ring atoms is a heteroatom selected from nitrogen, oxygen and sulfur and where one additional ring atom may be oxygen or sulfur. Examples of heterocyclic groups are furanyl, pyranyl, morpholinyl, dioxanyl and quinuclidinyl: 
Aryl refers to aromatic rings e.g., phenyl, substituted phenyl and the like, as well as rings which are fused, e.g., naphthyl, phenanthrenyl fluorenonyl and the like. An aryl group thus contains at least one ring having at least 6 atoms, with up to three such rings being present, containing up to 14 atoms therein, with alternating (resonating) double bonds between adjacent carbon atoms. The preferred aryl groups are phenyl, naphthyl, and fluorenone. Aryl groups may likewise be substituted as defined. Preferred substituted aryls include phenyl, fluorenonyl and naphthyl.
The term xe2x80x9cheteroarylxe2x80x9d (Het) refers to a monocyclic aromatic group having 5 or 6 ring atoms, a bicyclic aromatic group having 8 to 10 atoms, or tricyclic having 12-14 ring atoms, containing at least one heteroatom, O, S or N, in which a carbon or nitrogen atom is the point of attachment, and in which one or two additional carbon atoms is optionally replaced by a heteroatom selected from O or S, and in which from 1 to 3 additional carbon atoms are optionally replaced by nitrogen heteroatoms, said heteroaryl group being optionally substituted as described herein. Examples of this type are pyrrole, pyridine, oxazole, thiazole, dibenzofuran, dibenzothiophene, carbazole, phenanthrene, anthracene, dibenzothiophene sulfone, fluorenone, quinoline and oxazine. Additional nitrogen atoms may be present together with the first nitrogen and oxygen or sulfur, giving, e.g., thiadiazole. Examples include the following: 
Heteroarylium refers to heteroaryl groups bearing a quaternary nitrogen atom and thus a positive charge. Non-limiting examples include the following: 
When a charge is shown on a particular nitrogen atom in a ring which contains one or more additional nitrogen atoms, it is understood that the charge may reside on a different nitrogen atom in the ring by virtue of charge resonance that occurs. 
Similar charge resonance may occur in amidinium and guanidinium groups: 
The term xe2x80x9cheteroatomxe2x80x9d means O, S or N, selected on an independent basis.
Halogen and xe2x80x9chaloxe2x80x9d refer to bromine, chlorine, fluorine and iodine.
The term xe2x80x9cpro-drugxe2x80x9d refers to compounds with a removable group attached to one or both of the carboxyl groups of compounds of formula I (e.g. biolabile esters). Groups which are useful in forming pro-drugs should be apparent to the medicinal chemist from the teachings herein. Examples include pivaloyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl, and others described in detail in U.S. Pat. No. 4,479,947. These are also referred to as xe2x80x9cbiolabile estersxe2x80x9d.
The term xe2x80x9chydratexe2x80x9dis used in the conventional sense to include the compounds of formula I in physical association with water.
When a group is termed xe2x80x9csubstitutedxe2x80x9d, unless otherwise indicated, this means that the group contains from 1 to 3 substituents thereon.
A bond terminated by a wavy line is used herein to signify the point of attachment of a substituent group. This usage is illustrated by the following example: 
The terms xe2x80x9cquaternary nitrogenxe2x80x9d and xe2x80x9ccationic nitrogenxe2x80x9d refer to tetravalent, positively charged nitrogen atoms including, e.g., the positively charged nitrogen in a tetraalkylammonium group (e. g. xe2x80x94N+RuRvRw), heteroarylium, (e.g., N-methyl-imidazolium), amidinium, guanidinium, basic nitrogens which are protonated at physiological pH, and the like. A xe2x80x9ccationic groupxe2x80x9d is a moiety which contains at least one such quaternary nitrogen atom. Cationic groups thus encompass positively charged nitrogen-containing groups, as well as basic nitrogens which are protonated at physiologic pH. The terms dicationic, tricationic and tetracationic refer to groups which contain 2, 3 or 4 positively charged nitrogen atoms, respectively.
When a functional group is termed xe2x80x9cprotectedxe2x80x9d, this means that the group is in modified form to preclude undesired side reactions at the protected site. Suitable protecting groups for the compounds of the present invention will be recognized from the present application taking into account the level of skill in the art, and with reference to standard textbooks, such as Greene, T. W. et al. Protective Groups in Organic Synthesis Wiley, New York (1991). Examples of suitable protecting groups are contained throughout the specification.
In some of the compounds of the present invention suitable protecting groups represents hydroxyl-protecting, amine-protecting or carboxyl-protecting groups. Such conventional protecting groups consist of groups, which are used to protectively block the hydroxyl, amine or carboxyl group during the synthesis procedures described herein. These conventional blocking groups are readily removable, i.e., they can be removed, if desired, by procedures which will not cause cleavage or other disruption of the remaining portions of the molecule. Such procedures include chemical and enzymatic hydrolysis, treatment with chemical reducing or oxidizing agents under mild conditions, treatment with a transition metal catalyst and a nucleophile and catalytic hydrogenation.
Examples of carboxyl protecting groups include allyl, benzhydryl, 2-naphthylmethyl, benzyl, silyl such as t-butyldimethylsilyl (TBDMS), phenacyl, p-methoxybenzyl, o-nitrobenzyl, p-methoxyphenyl, p-nitrobenzyl, 4-pyridylmethyl and t-butyl.
Examples of suitable amine protecting groups include 9-fluorenylmethoxycarbonyl, p-nitrobenzyloxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, t-butyloxycarbonyl, 2,2,2-trichloroethyloxycarbonyl and the like.
Examples of suitable hydroxyl protecting groups include triethylsilyl, t-butyldimethylsilyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, t-butyloxycarbonyl, 2,2,2-trichloroethyloxycarbonyl and the like.
With respect to M1 and/or M2, this represents a carboxylic hydrogen, a carboxylate anion (M represents a negative charge), a pharmaceutically acceptable ester (M represents an ester forming group) or a pharmaceutically acceptable cation. When M1 and/or M2 is a negative charge it can be used to provide the necessary charge balance in a compound with one or more positive charges. Likewise, when M1 and/or M2 is a negative charge it can be balanced by the appropriate number of counterions, e.g., an alkali metal cation such as sodium or potassium. Other pharmaceutically acceptable counterions may be calcium, magnesium, zinc, ammonium, or alkylammonium cations such as tetramethylammonium, tetrabutylammonium, choline, triethylhydroammonium, meglumine, triethanolhydroammonium, etc.
For the purposes of this invention, all compounds have at least one Rq substituent containing at least one cationic nitrogen. Preferably 2 to 8 cationic nitrogens, more preferably 2 to 4 cationic nitrogens and most preferably 3 to 4 cationic nitrogens are present. The compounds are balanced with one or more, as necessary, of a charge balancing group Yxe2x88x92. Alternatively, the compounds can be balanced using M1 and/or M2 as the charge balancing group with or without the use of Yxe2x88x92. Examples of cases where a charge balancing group is required are quaternized substituents such as heteroarylium, xe2x80x94N+RuRvxe2x80x94E1xe2x80x94Q1, xe2x80x94N+RuRvRw, and the like. Additionally, all compounds having one or more anions are counter balanced with one or more, as necessary, charge balancing cations.
The compounds of the present invention are useful per se and in their pharmaceutically acceptable salt and ester forms are potentiators for the treatment of bacterial infections in animal and human subjects. The term xe2x80x9cpharmaceutically acceptable ester, salt or hydratexe2x80x9d, refers to those salts, esters and hydrated forms of the compounds of the present invention which would be apparent to the pharmaceutical chemist. For example, those which are substantially non-toxic and which may favorably affect the pharmacokinetic properties of said compounds, such as palatability, absorption, distribution, metabolism and excretion. Other factors, more practical in nature, which are also important in the selection, are cost of the raw materials, ease of crystallization, yield, stability, solubility, hygroscopicity and flowability of the resulting bulk drug. Conveniently, pharmaceutical compositions may be prepared from the active ingredients in combination with pharmaceutically acceptable carriers, Thus, the present invention is also concerned with pharmaceutical compositions and methods of treating bacterial infections utilizing as an active ingredient the novel carbapenemase compounds.
The pharmaceutically acceptable salts referred to above also include acid addition salts. Thus, the Formula I compounds can be used in the form of salts derived from inorganic or organic acids. Included among such salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.
Acid addition salts of the compounds of formula I include compounds that contain a protonated, basic moiety in Rq. Compounds containing a basic moiety in Rq are capable of protonation in aqueous media near pH 7, so that the basic moiety can exist as an equilibrium mixture of its neutral form and acid addition (protonated) form. The more basic the group, the greater the degree of protonation near pH 7. All such compounds are included in the present invention.
The pharmaceutically acceptable cations which can form a salt with one or both of the carboxyls (CO2M1 and CO2M2) of the compounds of formula I are known to those skilled in the art. Examples include those where M1 and M2 independently can be alkali metals such as sodium, potassium and the like, ammonium and the like.
The pharmaceutically acceptable esterifying groups are such as would be readily apparent to a medicinal chemist, and include, for example, those described in detail in U.S. Pat. No. 4,309,438. Included within such pharmaceutically acceptable esters are those which are hydrolyzed under physiological conditions, such as pivaloyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl, and others described in detail in U.S. Pat. No. 4,479,947. These are also referred to as xe2x80x9cbiolabile estersxe2x80x9d.
Biolabile esters are biologically hydrolizable, and may be suitable for oral administration, due to good absorption through the stomach or intestinal mucosa, resistance to gastric acid degradation and other factors. Examples of biolabile esters include compounds in which M1 and/or M2 represents an alkoxyalkyl, alkylcarbonyloxyalkyl, alkoxycarbonyloxyalkyl, cycloalkoxylalkyl, alkenyloxyalkyl, aryloxyalkyl, alkoxyaryl, alkylthioalkyl, cycloalkylthioalkyl, alkenylthioalkyl, arylthioalkyl or alkylthioaryl group. The following M1 and/or M2 species are examples of biolabile ester forming moieties: acetoxymethyl, 1-acetoxyethyl, 1-acetoxypropyl, pivaloyloxymethyl, 1-isopropyloxycarbonyloxyethyl, 1-cyclohexyloxycarbonyloxyethyl, phthalidyl and (2-oxo-5-methyl-1,3-dioxolen-4-yl)methyl.
Yxe2x88x92 can be present or absent as necessary to maintain the appropriate charge balance. When present, these represent pharmaceutically acceptable counterions. Most anions derived from inorganic or organic acids are suitable. Representative examples of such counterions are the following: acetate, adipate, aminosalicylate, anhydromethylenecitrate, ascorbate, aspartate, benzoate, benzenesulfonate, bromide, citrate, camphorate, camphorsulfonate, chloride, estolate, ethanesulfonate, fumarate, glucoheptanoate, gluconate, glutamate, lactobionate, malate, maleate, mandelate, methanesulfonate, pantothenate, pectinate, phosphate/diphosphate, polygalacturonate, propionate, salicylate, stearate, succinate, sulfate, tartrate and tosylate. Other suitable anionic species will be apparent to the ordinarily skilled chemist.
Likewise, when more than one negative charge is necessary to maintain charge neutrality, the counterion indicator may represent a specie with more than one negative charge, such as malonate, tartrate or ethylenediaminetetraacetate (EDTA), or two or more monovalent anions, such as chloride, etc. When a multivalent negatively charged counterion is present with a compound of formula I which bears a net single positive charge, an appropriate number of molar equivalents of the anionic species can be found in association therewith to maintain the overall charge balance and neutrality.
Some of the compounds of formula I may be crystallized or recrystallized from solvents such as organic solvents. In such cases solvates may be formed. This invention includes within its cope stoichiometric solvates including hydrates as well as compounds containing variable amounts of solvents such as water that may be produced by processes such as lyophilization. The compounds of formula I may be prepared in crystalline form by for example dissolution of the compound in water, preferably in the minimum quantity thereof, followed by admixing of this aqueous solution with a water miscible organic solvent such as a lower aliphatic ketone such as a di-(C1-6) alkyl ketone, or a (C1-6) alcohol, such as acetone or ethanol.
A subset of compounds of formula I which is of interest relates to those compounds where M1 and M2 are independently hydrogen or negative charge, said negative charge(s) balanced by the appropriate number of counter balancing ions, and all other variables are as described above.
Another subset of compounds of formula I which is of interest relates to those compounds where R1 and/or R2 represents a C1 to C16 straight, branched or unsaturated alkyl group substituted with 0 to 2 Rq, and substituted with 0 to 3 Rx groups and optionally interrupted by one of the following O, S, SO2, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94NRaxe2x80x94 and xe2x80x94CO2xe2x80x94, provided that at least one of R1 or R2 contains an Rq and all other variables are described as above.
Another subset of compounds of formula I which is of interest relates to those compounds where R1 and/or R2 represents C4-12 straight, branched or unsaturated alkyl group optionally substituted with 1-2 Rx and optionally substituted with 1-2 Rq groups, provided that at least one of R1 or R2 contains an Rq, wherein all other variables are as described above.
Another subset of compounds of formula I which is of interest relates to those compounds where R1 and/or R2 represents a group of the formula: 
wherein at least one Rq is present on R1 or R2 and all other variables are defined as above.
Another subset of compounds of formula I which is of interest relates to those compounds where R1 and/or R2 represents a group of the formula: 
wherein at least one Rq group is present on R1 or R2 and all other variables are defined as above.
Another subset of compounds of formula I which is of interest relates to those compounds where the relative and absolute stereochemistry is: 
Still another subset of compounds of formula I which is of interest relates to those compounds where R1 and/or R2 represents a group of the formula: 
wherein A is (CH2)1-5 and 
is phenyl, naphthyl, cyclohexyl or dibenzofuranyl, provided that at least one of R1 or R2 contains an Rq and all other variables are as originally defined.
Still another subset of compounds of formula I that is of interest relates to those compounds where R1 or R2 represents a group of the formula: 
wherein
A is (CH2)1-3, Axe2x80x2 is a single bond, xe2x80x94Oxe2x80x94 or (CH2)1-2 and 
xe2x80x83independently represent phenyl, thienyl, pyridyl, furanyl or cyclohexyl.
Yet another subset of compounds of formula I, that is of interest relates to those compounds where one of R1 or R2 is C4-8 straight, branched or unsaturated alkyl optionally substituted with 1 to 2 Rx or a group of the formula: 
where A is (CH2)1-2 and 
xe2x80x83is phenyl, cyclopentyl or cyclohexyl and the other of R1 or R2 is
i) a C7-12 alkyl group substituted with Rq,
ii) a group of the formula: 
xe2x80x83where A is (CH2)1-2, Axe2x80x2 is a single bond, 
is phenyl, thienyl or cyclohexyl and 
xe2x80x83is phenyl, thienyl or pyridyl, or
iii) a group of the formula: 
xe2x80x83where A is (CH2)1-3, 
xe2x80x83is phenyl or thienyl and Rq is xe2x80x94(CH2)2-6xe2x80x94Q+Yxe2x88x92 and all other variables are as originally defined.
Still another subset of compounds of formula I that is of interest relates to those compounds where:
R1 is C5-7 alkyl substituted with 0 to 2 Rx goups, 
R2 is C7-10 alkyl substituted with 1 Rq group and 0 to 2 Rx groups, 
xe2x80x83and all other variables are as described above.
Still another subset of compounds of formula I that is of interest relates to those compounds where:
R1 is: 
R2 is: 
xe2x80x83and all other variables are as described above.
A preferred subset of Rx is Ry.
It is preferred that a total of one or two Rq groups are present in R1 and R2 containing a total of 2 to 6 cationic nitrogen atoms. It is more preferred that a single Rq substituent is present containing a tricationic or tetracationic Q+ group. A more preferred Rq is xe2x80x94Exe2x80x94Q+Yxe2x88x92 wherein E is (CH2)0-6 or xe2x80x94C(O)xe2x80x94N(Ra)xe2x80x94(CH2)2-4xe2x80x94, and Q+ is a tricationic or tetracationic group.
Preferred tricationic Q+ groups are: 
wherein E1 is (CH2)2-4 or xe2x80x94(CH2)xe2x80x94C(O)xe2x80x94N(Ra)xe2x80x94(CH2)2-4xe2x80x94 and Ra, Q1 and Q2 are as previously defined.
More preferred tricationic Q+ groups are: 
wherein Ra, Ru, Rv, and Rw are independently selected as defined above.
Preferred tetracationic Q+ groups are: 
wherein E1 is (CH2)2-4 or xe2x80x94(CH2)xe2x80x94C(O)xe2x80x94N(Ra)xe2x80x94(CH2)2-4xe2x80x94 and Ra, Q1, Q2, and Rw are as defined above.
More preferred tetracationic Q+ groups are: 
wherein Ra, Ru, Rv, and Rw are as defined above.
Preferred Yxe2x88x92 groups are chloride, bromide, acetate, citrate, succinate, phosphate, maleate, tartrate and sulfate.
The compounds of the invention, which are succinic acids or derivatives thereof, can be formulated in pharmaceutical compositions by combining the compound with a pharmaceutically acceptable carrier. Examples of such carriers are set forth below. The compounds of formula I have metallo-xcex2-lactamase inhibitory properties, and are useful when combined with a xcex2-lactam antibiotic for the treatment of infections in animals, especially mammals, including humans. The compounds may be used, for example, in the treatment of infections of, amongst others, the respiratory tract, urinary tract and soft tissues and blood.
The compounds may be employed in powder or crystalline form, in liquid solution, or in suspension. They may be administered by a variety of means; those of principal interest include: topically, orally and parenterally by injection (intravenously or intramuscularly).
Compositions for injection, a preferred route of delivery, may be prepared in unit dosage form in ampules, or in multidose containers. The injectable compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain various formulating agents. Alternatively, the active ingredient may be in powder (lyophillized or non-lyophillized) form for reconstitution at the time of delivery with a suitable vehicle, such as sterile water. In injectable compositions, the carrier is typically comprised of sterile water, saline or another injectable liquid, e.g., peanut oil for intramuscular injections. Also, various buffering agents, preservatives and the like can be included.
Topical applications may be formulated in carriers such as hydrophobic or hydrophilic bases to form ointments, creams, lotions, in aqueous, oleaginous or alcoholic liquids to form paints or in dry diluents to form powders.
Oral compositions may take such forms as tablets, capsules, oral suspensions and oral solutions. The oral composions may utilize carriers such as conventional formulating agents, and may include sustained release properties as well as rapid delivery forms.
The compounds of the instant invention are metallo-xcex2-lactamase inhibitors, which are intended for use in pharmaceutical compositions. Accordingly, it is preferable that the metallo-xcex2-lactamase inhibitors are provided in substantially pure form, for example at least about 60% to about 75% pure, preferably about 85% to about 95% pure and most preferably about 98% or more pure (% are on a weight for weight basis). Impure preparations of the compounds may be used for preparing the more pure forms used in pharmaceutical compositions.
The dosage to be administered depends to a large extent upon the condition and size of the subject being treated, the route and frequency of administration, the sensitivity of the pathogen to the particular compound selected, the virulence of the infection and other factors. Such matters, however, are left to the routine discretion of the physician according to principles of treatment well known in the antibacterial arts. Another factor influencing the precise dosage regimen, apart from the nature of the infection and peculiar identity of the individual being treated, is the molecular weight of the compound.
The compositions for human delivery per unit dosage, whether liquid or solid, may contain from about 0.01% to as high as about 99% of active material, the preferred range being from about 10-60%. The composition will generally contain from about 15 mg to about 2.5 g of the active ingredient; however, in general, it is preferable to employ dosage amounts in the range of from about 250 mg to 1000 mg. In parenteral administration, the unit dosage will typically include the pure compound in sterile water solution or in the form of a soluble powder intended for solution, which can be adjusted to neutral pH and isotonic.
The invention described herein also includes a method of treating a bacterial infection in a mammal in need of such treatment comprising administering to said mammal a compound of formula I in conjunction with a xcex2-lactam antibiotic such as a carbapenem, penicillin or cephalosporin in an effective combination.
The preferred methods of administration of the Formula I compounds include oral and parenteral, e.g., i.v. infusion, i.v. bolus and i.m. injection.
The compounds of formula I may suitably be administered to the patient at a daily dosage of from 0.7 to 50 mg/kg of body weight. For an adult human (of approximately 70 kg body weight), from 50 to 3000 mg, preferably from 100 to 1000 mg, of a compound according to the invention may be administered daily, suitably in from 1 to 6, preferably from 2 to 4, separate doses. Higher or lower dosages may, however, be used in accordance with clinical practice.
The compounds may be used in combination with antibiotic agents for the treatment of infections caused by metallo-xcex2-lactamase producing strains, in addition to those infections which are subsumed within the antibacterial spectrum of the antibiotic agent. Metallo-xcex2-lactamase producing strains include: Bacillus cereus, Bacteroides fragilis, Aeromonas hydrophila, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, Stenotrophomonas maltophilia, Shigellaflexneri, Legionella gormanii, Chryseobacterium meningosepticum, Chryseobacterium indologenes, Acinetobacter baumannii, Citrobacterfreundii, and Aeromonas veronii. 
In accordance with the instant invention, it is generally advantageous to use a compound of formula I in admixture or conjuction with a carbapenem, penicillin, cephalosporin or other xcex2-lactam antibiotic or prodrug. It also advantageous to use a compound of formula I in combination with one or more xcex2-lactam antibiotics, because of the metallo-xcex2-lactamase inhibitory properties of the compounds. In this case, the compound of formula I and the xcex2-lactam antibiotic can be administered separately or in the form of a single composition containing both active ingredients.
Carbapenems, penicillins, cephalosporins and other xcex2-lactam antibiotics suitable for co-administration with the compounds of Formula I, whether by separate administration or by inclusion in the compositions according to the invention, include both those known to show instability to or to be otherwise susceptible to metallo-xcex2-lactamases and also known to have a degree of resistance to metallo-xcex2-lactamase.
When the compounds of Formula I are combined with antibiotics such as carbapenems dehydropeptidase (DHP) inhibitors may also be combined. Many carbapenems are susceptible to attack by a renal enzyme known as DHP. This attack or degradation may reduce the efficacy of the carbapenem antibacterial agent. Inhibitors of DHP and their use with carbapenems are disclosed in, e.g., (European Patent 0007614, filed Jul. 24, 1979 and application number 82107174.3, filed Aug. 9, 1982. A preferred DHP inhibitor is 7-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamide)-2-heptenoic acid or a useful salt thereof. Thus, compounds of the present invention in combination with a carbapenem such as imipenem and a DHP inhibitor such as, cilastatin is contemplated within the scope of this invention.
A serine xcex2-lactamase inhibitor such as clavulanic acid, sulbactam or tazobactam may also be co-administered with the compound of the invention and xcex2-lactam antibiotics, either by separate administration, or co-formulation with one, other or both of the compounds of the invention and the xcex2-lactam antibiotic.
Examples of carbapenems that may be co-administered with the compounds of formula I include imipenem, meropenem, biapenem, (4R,5S,6S)-3-[3S,5S)-5-(3-carboxyphenyl-carbamoyl)pyrrolidin-3-ylthio]-6-(1R)-1-hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, (1S,5R,6S)-2-(4-(2-(((carbamoylmethyl)-1,4-diazoniabicyclo[2.2.2]oct-1-yl)-ethyl(1,8-naphthosultam)methyl)-6-[1(R)-hydroxyethyl]-1-methylcarbapen-2-em-3-carboxylate chloride, BMS181139 ([4R-[4alpha,5beta,6beta(R*)]]-4-[2-[(aminoiminomethyl)amino]ethyl]-3-[(2-cyanoethyl)thio]-6-(1-hydroxyethyl)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid), BO2727 ([4R-3[3S*,5S*(R*)], 4alpha,5beta,6beta(R*)]]-6-(1-hydroxyethyl)-3-[[5-[1-hydroxy-3-(methylamino)propyl]-3-pyrrolidinyl]thio]-4-methyl-7-oxo-1-azabicyclo[3.2.0] hept-2-ene-2-carboxylic acid monohydrochloride), E1010 ((1R,5S,6S)-6-[1(R)-hydroxymethyl]-2-[2(S)-[1(R)-hydroxy-1-[pyrrolidin-3(R)-yl]methyl]pyrrolidin-4(S)-ylsulfanyl]-1-methyl-1-carba-2-penem-3-carboxylic acid hydrochloride), S4661((1R,5S,6S)-2-[(3S,5S)-5-(sulfamoylaminomethyl)pyrrolidin-3-yl]thio-6-[(1R)-1-hydroxyethyl]-1-methylcarbapen-2-em-3-carboxylic acid) and (1S,5R,6S)-1-methyl-2-{7-[4-(aminocarbonylmethyl)-1,4-diazoniabicyclo(2.2.2)octan-1yl]-methyl-fluoren-9-on-3-yl}-6-(1R-hydroxyethyl)-carbapen-2-em-3-carboxylate chloride.
Examples of penicillins suitable for co-administration with the compounds according to the invention include benzylpenicillin, phenoxymethylpenicillin, carbenicillin, azidocillin, propicillin, ampicillin, amoxycillin, epicillin, ticarcillin, cyclacillin, pirbenicillin, azloccillin, mezlocillin, sulbenicillin, piperacillin, and other known penicillins. The penicillins may be used in the form of pro-drugs thereof; for example as in vivo hydrolysable esters, for example the acetoxymethyl, pivaloyloxymethyl, xcex1-ethoxycarbonyloxy-ethyl and phthalidyl esters of ampicillin, benzylpenicillin and amoxycillin; as aldehyde or ketone adducts of penicillins containing a 6-xcex1-aminoacetamido side chain (for example hetacillin, metampicillin and analogous derivatives of amoxycillin); and as a-estsers of carbenicillin and ticarcillin, for example the phenyl and indanyl xcex1-esters.
Examples of cephalosporins that may be co-administered with the compounds according to the invention include, cefatrizine, cephaloridine, cephalothin, cefazolin, cephalexin, cephacetrile, cephapirin, cephamandole nafate, cephradine, 4-hydroxycephalexin, cephaloglycin, cefoperazone, cefsulodin, ceftazidime, cefuroxime, cefinetazole, cefotaxime, ceftriaxone, and other known cephalosporins, all of which may be used in the form of pro-drugs thereof.
Examples of xcex2-lactam antibiotics other than penicillins and cephalosporins that may be co-administered with the compounds according to the invention include aztreonam, latamoxef (Moxalactam-trade mark), and other known xcex2-lactam antibiotics such as carbapenems like imipenem, meropenem or (4R,5S,6S)-3-[(3S,5S)-5-(3-carboxyphenylcarbamoyl)pyrrolidin-3-ylthio]-6-(1R)-1-hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid, all of which may be used in the form of pro-drugs thereof.
Preferred carbapenems are imipenem, meropenem and (4R,5S,6S)-3-[(3S,5S)-5-(3-carboxyphenylcarbamoyl)pyrrolidin-3-ylthio]-6-(1R)-1-hydroxyethyl]-4-methyl-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid.
Particularly suitable penicillins for co-administration with the compounds according to the invention include ampicillin, amoxycillin, carbenicillin, piperacillin, azlocillin, mezlocillin, and ticarcillin. Such penicillins may be used in the form of their pharmaceutically acceptable salts, for example their sodium salts. Alternatively, ampicillin or amoxycillin may be used in the form of fine particles of the zwitterionic form (generally as ampicillin trihydrate or amoxycillin trihydrate) for use in an injectable or infusable suspension, for example, in the manner described herein in relation to the compounds of formula I. Amoxycillin, for example in the form of its sodium salt or the trihydrate, is particularly preferred for use in compositions according to the invention.
Particularly suitable cephalosporins for co-administration with the compounds according to the invention include cefotaxime, ceftriaxone and ceftazidime, which may be used in the form of their pharmaceutically acceptable salts, for example their sodium salts.
When the compositions according to this invention are presented in unit dosage form, each unit dose may suitably comprise from about 25 to about 1000 mg, preferably about from 50 to about 500 mg, of a compound according to the invention. Each unit dose may, for example, be 62.5, 100, 125, 150, 200 or 250 mg of a compound according to the invention.
When the compounds of formula I are co-administered with a penicillin, cephalosporin, carbapenem or other xcex2-lactam antibiotic, the ratio of the amount of the compounds of formula I to the amount of the other xcex2-lactam antibiotic may vary within a wide range. The said ratio may, for example, be from 100:1 to 1:100; more particularly, it may for example, be from 2:1 to 1:30. The amount of carbapenem, penicillin, cephalosporin or other xcex2-lactam antibiotic according to the invention will normally be approximately similar to the amount in which it is conventionally used.
The claimed invention also includes the use of a compound of formula I, a pharmaceutically acceptable salt, ester, prodrug, anhydride or solvate thereof, in the manufacture of a medicament for the treatment of bacterial infections.
The claimed invention also includes the use of a compound of formula I as a metallo-xcex2-lactamase inhibitor.
The claimed invention further includes a method of treating bacterial infections in humans or animals which comprises administering, in combination with a xcex2-lactam antibiotic, a therapeutically effective amount of a metallo-xcex2-lactamase inhibitor of formula I.
The claimed invention further includes a method of treating bacterial infections in humans or animals which comprises administering, in combination with a carbapenem antibiotic, a therapeutically effective amount of a metallo-xcex2-lactamase inhibitor of formula I.
The claimed invention also includes a composition comprising a metallo-xcex2-lactamase inhibitor of formula I together with a xcex2-lactam antibiotic and a pharmaceutically acceptable carrier.
The claimed invention also includes a composition comprising a metallo-xcex2-lactamase inhibitor of formula I together with a carbapenem antibiotic and a pharmaceutically acceptable carrier.
The compositions discussed above may optionally include a serine xcex2-lactamase inhibitor as described above as well as a DHP inhibitor.
Using standard susceptibility tests the compounds of the instant invention were found to be active against metallo-xcex2-lactamase enzymes produced by a range of organisms.
The compounds of the present invention are synthesized using the general conditions shown in the accompanying flow charts (A through F). 
The 2,3-disubstituted succinic acid compounds of the present invention can be prepared as described in Flow Sheets A-F. The cationic Rq substituents of the compounds of the present invention are generally carried through the syntheses in protected or precursory form and are then deprotected or elaborated near or at the end of the synthesis. Introduction of the Rq substituent from a precursor group is described in detail in Flow Sheet F.
The synthesis of Flow Sheet A is based on a known literature procedure (M. J. Crimmin et. al., SynLett 1993, 137). Referring to Flow Sheet A, the R1-substituted acetic acid starting materials A1 are readily available from commercial sources or are readily prepared by a variety of methods known in the art. Briefly, the starting material A1 is alkylated with an ester derivative of bromoacetic acid, employing a chiral auxiliary group to achieve stereoselectivity in the reaction. After removal of the chiral auxiliary to give A4, the R2* group is introduced stereoselectively by an alkylation reaction to give A5. The R2* group may be R2 as defined above, or may contain Rx and Rq substituents in precursory or protected form which require elaboration. Such elaboration may be carried-out at this point. Removal of the carboxyl protecting group of A5 then provides the final compound A6.
The first step of Flow Sheet A is introduction of the chiral auxiliary. A suggested method is as follows. A mixed anhydride is formed between the starting carboxylic acid A1 and pivalic acid by treating A1 with a tertiary amine base such as triethylamine and pivaloyl chloride in a suitable ethereal solvent such as tetrahydrofuran at reduced temperature such as between xe2x88x9278xc2x0 C. and 0xc2x0 C. After a suitable reaction time, such as from 30 min to 3 hours, the resulting activated intermediate is then reacted with a freshly prepared solution of lithio-(4R)-benzyl-2-oxazolidinone in tetrahydrofuran at reduced temperature such as between xe2x88x9278xc2x0 C. and 0xc2x0 C. After conventional isolation and purification, intermediate A2 is obtained. Intermediate A2 is deprotonated with a strong base such as sodium hexamethyldisilazide in a solvent such as tetrahydrofuran at reduced temperature such as between xe2x88x9278xc2x0 C. and xe2x88x9270xc2x0 C. The resulting enolate is alkylated by addition of BrCH2CO2P1, where P1 is a removable carboxyl protecting group. After an appropriate reaction period, such as from 1 to 3 hours, compound A3 is obtained by conventional isolation and purification techniques. Suitable removable ester derivatives of bromoacetic acid for this alkylation reaction are t-butyl bromoacetate, allyl bromoacetate or benzyl bromoacetate.
The oxazolidinone chiral auxiliary group of A3 is removed by a hydrolysis reaction. Aqueous lithium hydroxide and aqueous hydrogen peroxide are employed for this reaction along with an organic co-solvent such as tetrahydrofuran. The reaction is carried-out at a temperature of from 0xc2x0 C. to 30xc2x0 C. for a reaction time of from 30 min to 4 hours. After acidification, conventional isolation and purification provides intermediate A4.
An alternative method of removing the chiral auxilliary consists of reacting A3 with lithium benzyloxide (LiOCH2Ph) followed by cleavage of the resulting benzyl ester to give A4. The reaction of A3 with lithium benzyloxide is carried-out in tetrahydrofuran as solvent at a temperature of from xe2x88x9278xc2x0 C. to 30xc2x0 C. for a reaction time of from 30 min to 4 hours. Cleavage of the resulting benzyl ester is accomplished in conventional fashion, eg by hydrogenolysis employing a suitable catalyst such as palladium on carbon in an appropriate solvent such as ethanol at 1-2 atmospheres pressure of hydrogen. After conventional isolation and purification, compound A4 is obtained.
Alkylation of A4 to give A5 is accomplished by deprotonating A4 with  greater than 2 equivalents of a strong hindered base to give a dianion which is then reacted with an alkylating agent R2*L to give A5, where R2* is as defined above and L is a displaceable leaving group such as iodide, bromide or trifluoromethanesulfonate. The reaction proceeds with high stereoselectivity to give predominately the stereoisomer shown in Flow Sheet A. The deprotonation reaction is carried-out in a suitable solvent such as tetrahydrofuran at a temperature of from xe2x88x9278xc2x0 C. to xe2x88x9270xc2x0 C. for a reaction time of from 30 min to 3 hours. Preferred bases for this reaction are lithium bis(trimethylsilyl)amide and lithium diisopropylamide. After addition of the alkylating agent, the reaction is allowed to proceed at a temperature of from xe2x88x9278xc2x0 C. to 25xc2x0 C. for a reaction time of from 1 to 12 hours. Progress of the reaction can be monitored by conventional analytical methods, eg HPLC and TLC. Preferred alkylating agents for this reaction are alkyl iodides and alkyl bromides. Other suitable alkylating agents are well known in the art and include alkyl trifluoromethanesulfonates, alkyl methanesulfonates and alkyl tosylates. After conventional isolation and purification, intermediate A5 is obtained. The minor stereoisomer produced in this reaction can often be separated from A5 at this stage by conventional chromatographic techniques. However, it is often preferable to carry-out this separation at the stage of A6, after removal of the carboxyl protecting group as described below.
Deprotection of any Rx or Rq groups which are present in protected form may be accomplished at this point. For example, if compound A5 contains a protected hydroxyl or amino group, said protecting group may conveniently be removed at the stage of A5. Alternatively, depending on the nature of the protecting group it may be removed concurrent with or subsequent to the removal of the carboxyl protecting group as described immediately below. Introduction of the cationic Rq group may also be accomplished at this point from a precursor substituent. This procedure is described in detail in Flow Sheet F further below.
Removal of the carboxyl protecting group of A5 by standard methods gives the final compound A6. When P1 is t-butyl, this is accomplished by treating A5 with a strong acid such as trifluoroacetic acid in a suitable solvent such as dichloromethane. The reaction is carried-out at a temperature of from 0xc2x0 C. to 30xc2x0 C. for a reaction time of from 1 to 8 hours. The final compound A6 is then isolated by conventional techniques. Other methods of removing tert-butyl ester groups are known in the art and may also be employed (see e.g. Greene, T. W., et al. Protective Groups in Organic Synthesis, John Wiley and Sons. Inc., 1991).
It will be apparent to one skilled in the art that employing a chiral auxiliary of the opposite absolute configuration [e.g. lithio-(4S)-benzyl-2-oxazolidinone] in the first step of Flow Sheet A will make possible the synthesis of compound A3 with the alternative stereochemistry at the newly created stereocenter. This will in turn make possible the synthesis of the final compounds A6 of Flow Sheet A, with the opposite absolute configuration. Other chiral auxiliary groups are also known in the art and may also be employed.
Flow Sheet B illustrates a variation of Flow Sheet A which is preferred in certain cases, for example when Ar1 is a heteroaryl group such as pyridyl. In this synthesis the second substituent on the succinic acid is introduced by an aldol reaction instead of an alkylation reaction. The synthesis begins with compound A4, which is prepared as described in Flow Sheet A. Compound A4 is deprotonated with  greater than 2 equivalents of a strong hindered base to give a dianion which is then reacted with an aldehyde Ar1CHO to give B1, where Ar1 is an optionally substituted aryl or heteroaryl group, terms which are defined above. The deprotonation reaction is carried-out in a suitable solvent such as tetrahydrofuran at a temperature of from xe2x88x9278xc2x0 C. to xe2x88x9270xc2x0 C. for a reaction time of from 30 min to 3 hours. Preferred bases for this reaction are lithium bis(trimethylsilyl)amide and lithium diisopropylamide. After addition of the aldehyde, the reaction is allowed to proceed at a temperature of from xe2x88x9278xc2x0 C. to 25xc2x0 C. for a reaction time of from 1 to 12 hours. After conventional isolation and purification, intermediate B1 is obtained.
Compound B1 is next cyclized to the lactone B2. Suitable conditions for this cyclization reaction would be exposure of B1 to acetic anhydride and triethylamine in an inert solvent such as dichloromethane. Reductive opening of lactone B2, such as by hydrogenolysis over palladium on carbon in a suitable solvent such as methanol, provides compound B3. Deprotection of any Rx or Rq groups which are present in protected form may be accomplished at this point. In addition, introduction of a cationic Rq group from a precursor substituent may be carried-out at the stage of B3. This procedure is described in detail in Flow Sheet F further below. Removal of the carboxyl protecting group of B3 by conventional methods then gives the final compound B4.
Flow Sheet C illustrates an extension of the synthesis of Flow Sheet A which makes possible the introduction of a variety of preferred biaryl-type R2 substituents. Briefly, starting with compound A4 from Flow Sheet A, alkylation with Kxe2x80x94Ar2xe2x80x94(CH2)nxe2x80x94L by the method described in Flow Sheet A gives intermediate C1; where L is a displaceable leaving group such as iodide, bromide or trifluoromethanesulfonate, n is 1,2,3 or 4, Ar2 is an optionally substituted aryl or heteroaryl group as defined above, and K is iodide, bromide, chloride or a protected hydroxyl group which can be converted to a trifluoromethanesulfonate group by known methods. Protection of the free carboxyl group of C1 with a removable protecting group P2 gives C2. When K is a protected hydroxyl group it is deprotected and converted to a trifluoromethanesulfonate group at this point. A palladium catalyzed organometallic cross-coupling reaction between C2 and an organometallic reagent R3-Met gives compound C3; where Met is a boronic acid or trialkyltin moiety and R3 is an optionally substituted alkenyl, alkynyl, aryl or heteroaryl group as defined above. Deprotection or elaboration of any Rx or Rq groups which are present in protected or precursory form is accomplished at this point. Removal of the two carboxyl protecting groups of C3 then provides the final compound C4.
The P2 carboxyl protecting group is introduced in conventional fashion. A preferred P2 group is p-methoxybenzyl which can be introduced employing p-methoxybenzyl alcohol, a carbodiimide reagent such as 1,3-diisopropylcarbodiimide and N,N-dimethylaminopyridine catalyst in a suitable inert solvent such as dichloromethane. Other suitable ester protecting groups known in the art could also be employed (see e.g. Greene, T. W., et al. Protective Groups in Organic Synthesis, John Wiley and Sons. Inc., 1991).
The palladium catalyzed cross-coupling reaction between C2 and R3-Met is carried-out by procedures known in the scientific and patent literature. When Met is a boronic acid moiety [xe2x80x94B(OH)2] the reaction is commonly known as a Suzuki reaction (see Suzuki, Chem. Rev. 1995, 95, 2457). Compound C2 is combined with the boronic acid R3xe2x80x94B(OH)2 in a coupling solvent such as 1,2-dimethoxyethane, N,N-dimethylformamide or toluene, optionally with water as a co-solvent, with a base such as sodium carbonate and a palladium catalyst such as tetrakis(triphenylphosphine)-palladium(0). The reaction is carried-out at a temperature of from 20xc2x0 C. to 125xc2x0 C. for a reaction time of from 1 to 48 hours. The coupled product C3 is then isolated by conventional techniques. When Met is a trialkyltin moiety, the reaction is commonly known as a Stille reaction and the cross-coupling is carried-out by procedures well known in the literature (T. N. Mitchell, Synthesis 1992, 803).
Deprotection of any Rx or Rq groups which are present in protected form may be accomplished at this point. In addition, introduction of a cationic Rq group from a precursor substituent may be carried-out at the stage of C3. This procedure is described in detail in Flow Sheet F further below.
Removal of the carboxyl protecting groups of C3 by standard methods provides the final compound C4. It is often convenient for the protecting groups P1 and P2 to be selected such that they can both be removed under the same reaction conditions. For example, when P1 is tert-butyl and P2 is p-methoxybenzyl, both esters of C3 can be removed in a single step by treating C3 with a strong acid such as trifluoroacetic acid in a suitable solvent such as dichloromethane. It is sometimes advantageous to include a trapping agent such as triethylsilane or anisole in the reaction mixture. The reaction is carried-out at a temperature of from 0xc2x0 C. to 30xc2x0 C. for a reaction time of from 1 to 8 hours. The final compound C4 is then isolated by conventional techniques. Other methods of removing tert-butyl and p-methoxybenzyl ester groups are known in the art and may also be employed (see e.g. Greene, T. W., et al. Protective Groups in Organic Synthesis, John Wiley and Sons. Inc., 1991). Flow Sheet D illustrates an alternative synthesis of compounds of the present invention. The R1-substituted acetic acid starting materials D1 (M=H) and the esterified derivatives thereof (M=esterifying group) are readily available from commercial sources or are readily prepared by a variety of methods known in the art. The synthesis of Flow Sheet D is based on known literature procedures (see for example J. L. Belletire and D. F. Fry, J. Org. Chem. 1987, 52, 2549). Briefly, starting material D1 is deprotonated with a strong base and the resulting dianion (M=H) or anion (M=esterifying group) is oxidatively coupled with a suitable oxidizing reagent. In the case of M=H, acidic work-up and conventional isolation and purification gives the final compound D2. In the case of M=esterifying group, an additional deprotection step is also needed. A preferred strong base for the deprotonation reaction is lithium diisopropylamide. Suitable oxidizing agents for the synthesis of Flow Sheet D include iodine, copper(II) salts such as CuBr2, and titanium tetrachloride.
In the synthesis of Flow Sheet D, protection or elaboration of any Rx or Rq groups which are present in protected or precursory form is best accomplished where M=esterifying group, prior to removal of said esterifying group. In addition, introduction of a cationic Rq group from a precursor substituent may be carried-out as described in detail in Flow Sheet F further below.
Since the synthesis of Flow Sheet D is based on a dimerization-type reaction, it is best suited for the synthesis of symmetrically 2,3-disubstituted succinic acids (R1=R2). For this reason, it is generally less preferred than the syntheses of Flow Sheets A, B and C. The synthesis of Flow Sheet D also generally produces a racemic mixture of stereoisomers. However, it is possible to employ a chiral auxiliary in the synthesis of Flow Sheet D in order to achieve high stereoselectivity and optical purity (see for example N. Kise et. al. J. Org. Chem. 1995, 60, 1100). Such use of a chiral auxiliary is illustrated in Flow Sheet E.
Flow Sheet F illustrates a suggested method for the introduction of the cationic substituents of the compounds of the present invention from a precursor substituent. The starting material F1 of Flow Sheet F is substituted with a precursor substituent which can be elaborated into the desired cationic substitutent, Rq. A preferred precursor substituent is a protected hydroxymethyl group, P3OCH2xe2x80x94, where P3 is a removable hydroxyl protecting group. In Flow Sheet F, R4 is defined such that the moiety [xe2x80x94R4xe2x80x94CH2xe2x80x94Q+Yxe2x88x92] represents an R2 group as defined above. Examples of representative R4 groups are shown in Table 1.
The starting material F1 of Flow Sheet F is synthesized by one the methods described in Flow Sheets A, B, C, D, and E. When F1 is synthesized according to Flow Sheet A, it is derived from intermediate A5, through protection of the free carboxyl group with an appropriate carboxyl protecting group P2. In this case, the precursor substituent is present in the R1 or R2* substituent of A5. When F1 is synthesized according to Flow Sheet B, it is derived from intermediate B3, through protection of the free carboxyl group with an appropriate carboxyl protecting group P2. In this case, the precursor substituent is present in the R1 or Ar1 substituent of B3. When F1 is synthesized according to Flow Sheet C, it is derived from or corresponds to, intermediate C3. In this case, the precursor substituent is present in the R1, Ar2 or R3 substituent of C3. Starting material F1 may also be prepared by appropriate modification of the syntheses of Flow Sheets D and E as would be apparent to those skilled in the art.
Referring to Flow Sheet F, the first step is removal of the hydroxyl protecting group P3. This is accomplished by conventional methods. Hydroxyl protecting group P3 is generally selected such that it may be selectively removed in the presence of the carboxyl protecting groups P1 and P2. A preferred P3 is t-butyldimethylsilyl. Removal of the preferred t-butyldimethylsilyl P3 is accomplished by treating F1 with tetra-n-butylammonium fluoride and acetic acid in tetrahydrofuran as solvent. Other hydroxyl protecting groups are well known in the art and may also be employed (see e.g. Greene, T. W., et al. Protective Groups in Organic Synthesis, John Wiley and Sons. Inc., 1991).
Introduction of the cationic substituent is accomplished by an activation-dispacement process. Briefly, the hydroxyl group of F2 is converted into a suitable leaving group, G, which is thereafter displaced with a nucleophilic nitrogen compound Q*, to yield F4. With certain Q* groups, additional steps may also be needed such as removal of amino protecting groups or conversion of an amine precursor such azide into an amino group. The protecting groups are removed from F4 in conventional fashion and then in an optional step a pharmaceutically acceptable counterion Yxe2x88x92 may be introduced to provide compound F5.
The following are examples of suitable leaving groups G: alkyl and substituted alkylsulfonates, aryl and substituted arylsulfonates and halides. The common sulfonate leaving groups are: methanesulfonyloxy, trifluoromethanesulfonyloxy, fluorosulfonyloxy, p-toluenesulfonyloxy, and 2,4,6-triisopropylbenzenesulfonyloxy. The preferred halogen leaving groups are bromide and iodide.
Compound Q* represents a precursor group to the cationic group Q+ as defined above. As such, it may require further modification after its reaction with F3. The nucleophilic nitrogen moiety of Q* is generally the nitrogen of a primary, secondary or tertiary amino group or a ring nitrogen of a heteroaryl group such as a 1-substituted-imidazole. In addition to its nucleophilic nitrogen atom, Q* may include 1,2 or 3 of the following moieties: positively charged nitrogen atoms, protected amino groups, amine precursor groups such as azido. Suitable protecting groups for amino groups present in Q* would be t-butyloxycarbonyl-, allyloxycarbonyl- and p-nitrobenzyloxycarbonyl-. The Q* groups may be prepared by standard methods known in the scientific and patent literature. Suitable Q* groups are listed in Table 2.
Referring to Flow Sheet F, the hydroxyl group of F2 may be converted into a suitable alkyl- or arylsulfonate leaving group by treating with an appropriate agent such as an alkyl- or arylsulfonyl chloride or an alkyl- or arylsulfonic anhydride in the presence of a hindered organic base such as triethylamine or 2,6-lutidine. A suitable solvent such as dichloromethane is employed and the reaction is carried out at reduced temperature, such as from about xe2x88x9270xc2x0 C. to 0xc2x0 C.
The preferred halogen leaving groups may be introduced by displacing an alkyl- or arylsulfonate leaving group with an appropriate metal halide. Thus, compound F3, where G is an alkyl- or arylsulfonate group, is reacted with a suitable metal halide such as sodium iodide or potassium bromide in a suitable solvent such as acetone, acetonitrile, tetrahydrofuran, 1-methyl-2-pyrrolidinone and the like, at from about 0xc2x0 C. to 50xc2x0 C. Alternatively, the hydroxyl group of F2 may be directly converted into an iodide group by reaction with an appropriate reagent, e.g. by treatment of F2 with methyl triphenoxyphosphonium iodide in a suitable solvent, such as N,N-dimethylformamide, at reduced or ambient temperatures. Introduction of the cationic substituent is accomplished by reacting F3 with a nucleophilic nitrogen compound Q* in a suitable solvent, such as acetonitrile, tetrahydrofuran, 1-methyl-2-pyrrolidinone and the like, at about 0xc2x0 C. to 50xc2x0 C. to provide F4. When the leaving group, G, is iodide or bromide, this displacement reaction may also be facilitated by the addition of silver trifluoromethanesulfonate to the reaction mixture.
When the hydroxyl group of F2 is located at a benzylic position, and the reactive trifluoromethanesulfonate group is employed as the leaving group G in F3, the activation and displacement steps must be carried-out in situ, since in this case F3 cannot be isolated by conventional techniques due to its instability. Thus, treatment of F2 with a slight excess of trifluoromethanesulfonic anhydride in the presence of a hindered, non-nucleophilic base such as 2,6-lutidine, 2,4,6-collidine, or 2,6-di-tert-butyl-4-methyl-pyridine in a suitable solvent, such as dichloromethane or acetonitrile, at from about xe2x88x9278xc2x0 C. to xe2x88x9220xc2x0 C. provides for the generation of the trifluoromethanesulfonate activating group. Introduction of the cationic group is then accomplished by reacting the above trifluoromethanesulfonate intermediate in situ with Q* at reduced temperature. It is also possible in certain instances to use the nucleophilic nitrogen compound Q* as the base for the formation of the trifluoromethanesulfonate activating group. In this case, treatment of F2 with trifluoromethanesulfonic anhydride in the presence of at least two equivalents of Q* at reduced temperature such as from xe2x88x9278xc2x0 C. to 0xc2x0 C. provides intermediate F4. Examples of Q* which are suitable for use in this manner are 1-methylimidazole and 1,4-diazabicyclo(2.2.2)octane.
Removal of the carboxyl protecting groups of F4 by standard methods provides the final compound F5. If Q* includes one or more protected amino groups, these are removed either before, after or simultaneous with the carboxyl protecting groups depending on the exact nature of the protecting groups. If Q* includes one or more amine precursor groups, these may be converted to the desired amine or amines either before or after removal of the carboxyl protecting groups depending on the nature of the protecting groups. In the case of an azido amine precursor group, this may be accomplished by hydrogenation over a suitable catalyst such as rhodium on carbon. After the protecting groups are removed from F4, and the cationic group Q+ has been fully elaborated, the final compound F5 is isolated by conventional techniques. As an optional final step, a pharmaceutically acceptable counterion Yxe2x88x92, which may differ from Gxe2x88x92, may be introduced by standard techniques, e.g. by employing an anion exchange resin. Suitable negatively charged counterions are listed above under the description of pharmaceutically acceptable salts.
Compound F5 is electronically balanced. If more than one positive charge is present in the cationic Q+ group of F5, it is understood that an appropriate amount of negative counterion is Yxe2x88x92 present to result in overall electronic balance in the final compound F5. Likewise, it is understood that when the counterion Yxe2x88x92 is an anionic species possessing more than one negative charge, then an appropriate amount of Yxe2x88x92 is present to result in overall electronic balance in the final compound of Formula I. For example, when Yxe2x88x92 is a dianionic species, then one-half of a molar equivalent of Yxe2x88x92 is present relative to the succinate moiety.
Representative examples of R4 and Q* are found below in Tables 1 and 2 respectively: